Das E. coli Expressionssystem ist das am häufigsten angewandte hinsichtlich der rekombinante Proteinexpression für strukturelle und funktionelle Analysen aufgrund der hohen erzielten Ausbeuten und der einfachen Handhabbarkeit. Allerdings ist insbesondere die Expression eukaryotischer Proteine in E. coli problematisch, z.B. wenn das Protein nicht korrekt gefaltet ist und in unlöslichen Inclusion Bodies anfällt. In manchen Fällen ist die Analyse von Deletionskonstrukten oder einzelnen Proteindomänen der Untersuchung des Vollängeproteins vorzuziehen. Dies umfasst die Herstellung eines Satzes von Expressionskonstrukten, welche charakterisiert werden müssen. In dieser Arbeit werden Methoden optimiert und evaluiert für die in vitro-Faltung von Inclusion Body-Proteinen sowie die Entwicklung einer Hochdurchsatz-Charakterisierung von Expressionskonstrukten. Die Überführung von Inclusion Body-Proteinen in den nativen Zustand beinhaltet zwei Schritte: (a) Auflösen mit einen chaotropen Reagenz oder starkem ionischen Detergenz und (b) Faltung des Proteins durch Beseitigung des Chaotrops begleitet von dem Transfer in einen geeigneten Puffer. Die Ausbeute an nativ gefaltetem Protein ist oft stark eingeschränkt aufgrund von Aggregation und Fehlfaltung; sie kann allerdings durch die Zugabe bestimmter Additive zum Faltungspuffer erhöht werden. Solche Additive müssen empirisch identifiziert werden. In dieser Arbeit wurde eine Testprozedur für Faltungsbedingungen entwickelt. Zur Reduzierung der möglichen Kombinationen der getesteten Additive wurden sowohl empirische Beobachtungen aus der Literatur als auch bekannte Eigenschaften der Additive berücksichtigt. Zur Verminderung der eingesetzten Proteinmenge und des Arbeitsaufwandes wurde der Test automatisiert und miniaturisiert mittels eines Pipettierroboters. 20 Bedingungen zum schnellen Verdünnen von denaturierten Proteinen werden hierbei getestet und zwei Bedingungen zur Faltung von Proteinen mit dem Detergenz/Cyclodextrin Protein-Faltungssystem von Rozema et al. (1996). 100 µg Protein werden pro Bedingung eingesetzt. Zusätzlich werden acht Bedingungen für die Faltung von His-Tag-Fusionsproteinen (ca. 200 µg), welche an eine Metallchelat-Matrix immobilisiert sind, getestet. Die Testprozedur wurde erfolgreich angewendet zur Faltung eines humanen Proteins, der p22 Untereinheit von Dynactin, welche in E. coli in Inclusion Bodies exprimiert wird. So wie es sich bei vielen Proteinen darstellt, war auch für p22 Dynactin kein biologischer Nachweistest vorhanden, um den Erfolg des Faltungsexperimentes zu messen. Die Löslichkeit des Proteins kann nicht als eindeutiges Kriterium dienen, da neben nativ gefaltetem Protein, lösliche fehlgefaltete Spezies und Mikroaggregate auftreten können. Diese Arbeit evaluiert Methoden zur Detektion kleiner Mengen nativen Proteins nach dem automatisierten Faltungstest. Bevor p22 Dynactin gefaltet wurde, wurden zwei Modellenzyme zur Evaluierung eingesetzt, bovine Carboanhydrase II (CAB) und Malat Dehydrogenase aus Schweineherz-Mitochondrien. Die wiedererlangte Aktivität nach der Rückfaltung wurde korreliert mit verschiedenen biophysikalischen Methoden. Bindungsstudien mit 8-Anilino-1-Naphtalenesulfonsäure ergaben keine brauchbaren Informationen bei der Rückfaltung von CAB aufgrund der zu geringen Sensitivität und da fehlgefaltete Proteine nicht eindeutig von nativem Protein unterschieden werden konnten. Tryptophan Fluoreszenzspektren der rückgefalteten CAB wurden zur Einschätzung des Erfolges der Rückfaltung angewandt. Die Verschiebung des Intensitätsmaximum zu einer niedrigeren Wellenlänge im Vergleich zum denaturiert entfalteten Protein sowie die Fluoreszenzintensität korrelierten mit der wiedererlangten enzymatischen Aktivität. Für beide Modellenzyme war analytische hydrophobe Interaktionschromatographie (HIC) brauchbar zur Identifizierung rückgefalteter Proben mit aktivem Enzym. Kompakt gefaltetes, aktives Enzym eluierte in einem distinkten Peak im abnehmenden Ammoniumsulfat-Gradienten. Das Detektionslimit für analytische HIC lag bei 5 µg. Im Falle von CAB konnte gezeigt werden, dass Tryptophan-Fluoreszenz-Spektroskopie und analytische HIC in Kombination geeignet sind um Falsch-Positive oder Falsch-Negative, welche mit einem der Monitore erhalten wurden, auszuschließen. Diese beiden Methoden waren ebenfalls geeignet zur Identifizierung der Faltungsbedingungen von p22 Dynactin. Tryptophan-Fluoreszenz-Spektroskopie kann jedoch zu Falsch-Positiven führen, da in machen Fällen Spektren von löslichen Mikroaggregaten kaum unterscheidbar sind von Spektren des nativ gefalteten Proteins. Dies zusammenfassend wurde eine schnelle und zuverlässige Testprozedur entwickelt, um Inclusion Body-Proteine einer strukturellen und funktionellen Analyse zugänglich zu machen. In einem separaten Projekt wurden 88 verschiedene E. coli-Expressionskonstrukte für 17 humane Proteindomänen, welche durch Sequenzanalyse identifiziert wurden, mit einer Hochdurchsatzreinigung und –faltungsanalytik untersucht, um für die Strukturanalyse geeignete Kandidaten zu erhalten. Nach Expression in einem Milliliter im 96er Mikrotiterplattenformat und automatisierter Proteinreinigung wurden löslich exprimierte Proteindomänen direkt analysiert mittels 1D ¹H-NMR Spektroskopie. Hierbei zeigte sich, dass insbesondere isolierte Methylgruppen-Signale unter 0.5 ppm sensitive und zuverlässige Sonden sind für gefaltetes Protein. Zusätzlich zeigte sich, dass – ähnlich zur Evaluierung des Faltungstests – analytische HIC effizient eingesetzt werden kann zur Identifizierung von Konstrukten, welche kompakt gefaltetes Protein ergeben. Sechs Konstrukte, welche zwei Domänen repräsentieren, konnten schnell als tauglich für die Strukturanalyse gefunden werden. Die Struktur einer dieser Domänen wurde kürzlich von Mitarbeitern gelöst, die andere Struktur wurde im Laufe dieses Projektes von einer anderen Gruppe veröffentlicht. / For recombinant production of proteins for structural and functional analyses, the E. coli expression system is the most widely used due to high yields and straightforward processing. However, particularly the expression of eukaryotic proteins in E. coli is often problematic, e.g. when the protein is not folded correctly and is deposited in insoluble inclusion bodies. In some cases it is favourable to analyse deletion constructs of a protein or an individual protein domain instead of the full-length protein. This implies the generation of a set of expression constructs that need to be characterised. In this work methods to optimise and evaluate in vitro folding of inclusion body proteins as well as high-throughput characterisation of expression constructs were developed. Transferring inclusion body proteins to their native state involves two steps: (a) solubilisation with a chaotropic reagent or a strong ionic detergent and (b) folding of the protein by removal of the chaotrop accompanied by the transfer into an appropriate buffer. The yield of natively folded protein is often substantially reduced due to aggregation or misfolding; it may, however, be improved by certain additives to the folding buffer. These additives need to be identified empirically. In this thesis a screening procedure for folding conditions was developed. To reduce the number of possible combinations of screening additives, empirical observations documented in the literature as well as well known properties of certain screening additives were considered. To decrease the amount of protein and work invested, the screen was miniaturised and automated using a pipetting robot. Twenty rapid dilution conditions for the denatured protein are tested and two conditions for folding of proteins using the detergent/cyclodextrin protein folding system of Rozema et al. (1996). 100 µg protein is used per condition. In addition, eight conditions can be tested for folding of His-tagged proteins (approx. 200 µg) immobilised on metal chelate resins. The screen was successfully applied to fold a human protein, the p22 subunit of dynactin that is expressed in inclusion bodies in E. coli. For p22 dynactin – as is the case for many proteins – there was no biological assay available to assess the success of the folding screen. Protein solubility can not be used as a stringent criterion because beside natively folded protein, soluble misfolded species and microaggregates may occur. This work evaluates methods to detect small amounts of natively folded protein after automated folding screening. Before folding screening with p22 dynactin, two model enzymes, bovine carbonic anhydrase II (CAB) and pig heart mitochondrial malate dehydrogenase, were used for evaluation. Recovered activity after refolding was correlated to different biophysical methods. 8-anilino-1-naphtalenesulfonic acid binding-experiments gave no useful information when refolding CAB, due to low sensitivity and because misfolded protein could not be readily distinguished from native protein. Tryptophan fluorescence spectra of refolded CAB were used to assess the success of refolding. The shift of the intensity maximum to a shorter wavelength, compared to the denaturant unfolded protein, as well as the fluorescence intensity correlated to recovered enzymatic activity. For both model enzymes, analytical hydrophobic interaction chromatography (HIC) was useful to identify refolded samples that contain active enzyme. Compactly folded, active enzyme eluted in a distinct peak in a decreasing ammonium sulfate gradient. The detection limit of analytical HIC was approx. 5 µg. In case of CAB, tryptophan fluorescence spectroscopy and analytical HIC showed that both methods in combination can be useful to rule out false positives or false negatives obtained with one method. These two methods were also useful to identify conditions for folding of p22 dynactin. However, tryptophan fluorescence spectroscopy can lead to false positives because in some cases spectra of soluble microaggregates are not well distinguishable from spectra of natively folded protein. In summary, a fast and reliable screening procedure was developed to make inclusion body proteins accessible to structural or functional analyses. In a separate project, 88 different E. coli expression constructs for 17 human protein domains that had been identified by sequence analysis were analysed using high-throughput purification and folding analysis in order to obtain candidates suitable for structural analysis. After 96 deep-well microplate expression and automated protein purification, solubly expressed protein domains were directly analysed using 1D ¹H-NMR spectroscopy. It was found that isolated methyl group signals below 0.5 ppm are particularly sensitive and reliable probes for folded protein. In addition – similar to the evaluation of a folding screen – analytical HIC proved to be an efficient tool for identifying constructs that yield compactly folded protein. Both methods, 1D ¹H-NMR spectroscopy and analytical HIC, provided complementary results. Six constructs, representing two domains, could be quickly identified as targets that are well suitable for structural analysis. The structure of one of these domains was solved recently by co-workers, the other structure was published by another group during this project.

Investigations into the origin and evolution of regulatory mechanisms require quantitative estimates of the abundance and co-occurrence of functional protein domains among distantly related genomes. The metabolic and regulatory capabilities of an organism are implicit in its protein content. Currently available methods suffer for strong ascertainment biases, requiring methods for unbiased approaches to protein domain contents at genome-wide scales. The discussion will be highlighted on large scale patterns of similarities and differences of domain contains between phylum-level or even higher level taxonomic groups. This provides insights into large-scale evolutionary trends. The complement of recognizable functional protein domains and their combinations convey essentially the same information and at the same time are much more readily accessible, although protein domain models trained for one phylogenetic group frequently fail on distantly related sequences. Transcription factors (TF) typically cooperate to activate or repress the expression of genes. They play a critical role in developmental processes. While Chromatin Regulation (CR) facilitates DNA organization and prevent DNA aggregation and tangling which is important for replication, segregation, and gene expression. To compare the set of TFs and CRs between species, the genome annotation of equal quality was employed. However, the existing annotation suffers from bias in model organism. The similar count of transcripts are expected to be similar in mammals, but model organism such as human has more annotated transcripts than non model such as gorilla. Moreover, closely related species (e.g, dolphin and human) show a dramatically different distribution of TFs and CRs. Within vertebrates, this is unreasonable and contradicts phylogenetic knowledge. To overcome this problem, performing gene prediction followed by the detection of functional domains via HMM-based annotation of SCOP domains were proposed. This methods was demonstrated to lead toward consistent estimates for quantitative comparison. To emphasize the applicability, the protein domain distribution of putative TFs and CRs by quantitative and boolean means were analyzed. In particular, systematic studies of protein domain occurrences and co-occurrences to study avoidance or preferential co-occurrence of certain protein domains within TFs and CRs were utilized. Pooling related domain models based on their GO-annotation in combination with de novo gene prediction methods provides estimates that seem to be less affected by phylogenetic biases. it was shown for 18 diverse representatives from all eukaryotic kingdoms that a pooled analysis of the tendencies for co-occurrence or avoidance of protein domains is indeed feasible. This type of analysis can reveal general large-scale patterns in the domain co-occurrence and helps to identify lineage-specific variations in the evolution of protein domains. Somewhat surprisingly, strong ubiquitous patterns governing the evolutionary behavior of specific functional classes were not found. Instead, there are strong variations between the major groups of Eukaryotes, pointing at systematic differences in their evolutionary constraints. Species-specific training is required, however, to account for the genomic peculiarities in many lineages. In contrast to earlier studies wide-spread statistically significant avoidance of protein domains associated with distinct functional high-level gene-ontology terms were found.

Investigations into the origin and evolution of regulatory mechanisms require quantitative estimates of the abundance and co-occurrence of functional protein domains among distantly related genomes. The metabolic and regulatory capabilities of an organism are implicit in its protein content. Currently available methods suffer for strong ascertainment biases, requiring methods for unbiased approaches to protein domain contents at genome-wide scales. The discussion will be highlighted on large scale patterns of similarities and differences of domain contains between phylum-level or even higher level taxonomic groups. This provides insights into large-scale evolutionary trends. The complement of recognizable functional protein domains and their combinations convey essentially the same information and at the same time are much more readily accessible, although protein domain models trained for one phylogenetic group frequently fail on distantly related sequences. Transcription factors (TF) typically cooperate to activate or repress the expression of genes. They play a critical role in developmental processes. While Chromatin Regulation (CR) facilitates DNA organization and prevent DNA aggregation and tangling which is important for replication, segregation, and gene expression. To compare the set of TFs and CRs between species, the genome annotation of equal quality was employed. However, the existing annotation suffers from bias in model organism. The similar count of transcripts are expected to be similar in mammals, but model organism such as human has more annotated transcripts than non model such as gorilla. Moreover, closely related species (e.g, dolphin and human) show a dramatically different distribution of TFs and CRs. Within vertebrates, this is unreasonable and contradicts phylogenetic knowledge. To overcome this problem, performing gene prediction followed by the detection of functional domains via HMM-based annotation of SCOP domains were proposed. This methods was demonstrated to lead toward consistent estimates for quantitative comparison. To emphasize the applicability, the protein domain distribution of putative TFs and CRs by quantitative and boolean means were analyzed. In particular, systematic studies of protein domain occurrences and co-occurrences to study avoidance or preferential co-occurrence of certain protein domains within TFs and CRs were utilized. Pooling related domain models based on their GO-annotation in combination with de novo gene prediction methods provides estimates that seem to be less affected by phylogenetic biases. it was shown for 18 diverse representatives from all eukaryotic kingdoms that a pooled analysis of the tendencies for co-occurrence or avoidance of protein domains is indeed feasible. This type of analysis can reveal general large-scale patterns in the domain co-occurrence and helps to identify lineage-specific variations in the evolution of protein domains. Somewhat surprisingly, strong ubiquitous patterns governing the evolutionary behavior of specific functional classes were not found. Instead, there are strong variations between the major groups of Eukaryotes, pointing at systematic differences in their evolutionary constraints. Species-specific training is required, however, to account for the genomic peculiarities in many lineages. In contrast to earlier studies wide-spread statistically significant avoidance of protein domains associated with distinct functional high-level gene-ontology terms were found.

Der Transkriptionsfaktor CCAAT/enhancer-binding Protein β (C/EBPβ) reguliert die Expression zahlreicher Gene, welche die Proliferation, Differenzierung und Seneszenz in hämatopoietischen Zellen, Adipozyten und Leukämiezellen kontrollieren. Um diese mannigfaltigen Aufgaben zu erfüllen interagiert C/EBPβ mit zahlreichen Kofaktoren und Proteinen der Transkriptionsregulations-Maschinerie. Da das funktionale Netzwerk von C/EBPβ und seinen zahlreichen Kooperationspartnern bis heute nicht vollständig entziffert ist, ist es das Ziel dieser Arbeit das Netzwerk aus Interaktionspartnern und C/EBPβ regulierten Proteinen in Leukämiezelllinien und darüber hinaus zu erforschen und aufzudecken. Das Interaktom von C/EBPβ wurde mittels einer Kombination aus einem membranbasierten Peptid-Interaktions Testverfahrens (APS) und endogener Immunprezipitationen mit gekoppelter MS-Analyse untersucht. Außerdem wurde die Proteinmenge von C/EBPβ und von potentiell von C/EBPβ regulierten Proteinen mittels proteomischer MS-Analyse in C/EBPβ Knock-out- und Leukämiezelllinien untersucht. Die Protein-Interaktionsversuche ergaben epigenetische und allgemeine transkriptionsregulierende Proteine, sowie Chromatinstruktur modellierende Faktoren, die mit C/EBPβ interagieren. Zusätzlich konnten neue Interaktionen von C/EBPβ mit Kondensin- und Kinetochorproteinen beobachtet werden. Die Versuchsergebnisse eröffnen überdies neue Interaktionen von C/EBPβ mit DNA Reparatur und Apoptose assoziierten Proteinen. Interessanterweise konnten auch Komponenten des Spliceosomes und RNA-prozessierende Proteine als Interaktoren von C/EBPβ identifiziert werden. Zusammenfassend ermöglicht diese Studie nicht nur die Verifikation von bereits bekannten Proteininteraktionen von C/EBPβ, sondern eröffnet zahlreiche weitere zukünftige Forschungsfelder bezüglich des Interaktionsnetzwerkes von C/EBPβ in Leukämien, sowie anderen Zellarten und Geweben. / The basic leucine zipper transcription factor CCAAT/enhancer-binding protein β (C/EBPβ) regulates the expression of various genes that control the proliferation, differentiation and senescence of haematopoietic cells, adipocytes and leukemia cells. To facilitate its multifaceted functions C/EBPβ interacts with a collection of cofactors and proteins of the transcription regulation machinery. As the functional network of C/EBPβ and its numerous cooperation partners is still incomplete this study attempted to analyze interaction partners and downstream proteins of C/EBPβ in leukemia cells and beyond. A combinatory approach of an array based peptide-interaction screening (APS) and endogenous shotgun IP-MS from leukemia cell lines was applied to elucidate the interactome of C/EBPβ. Moreover, C/EBPβ abundance and potential C/EBPβ regulated proteins were determined by MS proteomics in C/EBPβ knockout and leukemia cell lines. The interaction screenings revealed proteins associated with the general and epigenetic regulation of transcription, with chromatin remodeling and mitotic chromatin organization as well as cell cycle regulation. Additionally, new interactions of C/EBPβ with condensin and kinetochore proteins could be elucidated. The data reports of novel C/EBPβ interactors involved in DNA repair and apoptosis. In addition, components of the spliceosome and RNA-processing were detected. Altogether this study verifies known and reveals various novel interactions of the transcription factor C/EBPβ and augments the network of previous reported interactions and potential cooperation partners. The here collected data discloses new subjects for further research concerning the interaction network of C/EBPβ during cell differentiation and in leukemia.